The capacity to introduce a particular foreign or native gene sequence into a cell and to control the expression of that gene is of value in the fields of medicine and biological research. Such capacity has a wide variety of useful applications, including but not limited to studying gene regulation and designing a therapeutic basis for the treatment of disease.
The introduction of a particular foreign or native gene into a host cell can be facilitated by introducing a gene sequence into a suitable nucleic acid vector. A variety of methods have been developed that allow the introduction of such a recombinant vector into a desired host cell. The use of viral vectors can result in the rapid introduction of the recombinant molecule into a wide variety of host cells.
Retroviruses are RNA viruses that replicate through a DNA proviral intermediate that is usually integrated in the genome of the infected host cell. All known retroviruses share features of the replicative cycle, including packaging of viral RNA into virions, entry into target cells, reverse transcription of viral RNA to form the DNA provirus, and stable integration of the provirus into the target cell genome. Replication competent simple proviruses typically comprise regulatory long terminal repeats (LTRs) and the gag, pro, pot and env genes which encode core proteins (gag), a protease (pro), reverse transcriptase (pol), RNAse H (pol), integrase (pol) and envelope glycoproteins (env). Complex retroviruses also typically comprise additional accessory genes.
Retroviral vectors are a common tool for gene delivery in that the ability of retroviral vectors to deliver an unrearranged, single copy gene into a broad range of cells makes them well suited for transferring genes to a cell. While recombinant retroviral vectors allow for integration of a transgene into a host cell genome, most retroviruses can only transduce dividing cells. This can limit their use for in vivo gene transfer to non-proliferating cells such as hepatocytes, myofibers, hematopoietic stem cells (HSCs), and neurons. Non-dividing cells are the predominant, long-lived cell type in the body, and account for most desirable targets of gene transfer, including liver, muscle, and brain.
Lentiviruses are a subgroup of retroviruses that are capable of infecting non-dividing cells. These viruses include, but are not limited to, HIV-1, HIV-2, SIV, EIAV, and FIV. Lentiviruses possess gag, pol, and env genes in addition to other accessory genes that are flanked by two long terminal repeat (LTR) sequences.
A key challenge for gene transfer based on the use of retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis and induction of the DNA damage response due to the presence of double stranded DNA. One approach to avoid insertional mutagenesis is to target the transgene integration to a specific location on the genome.
One approach to achieving transgene expression while minimizing insertional mutagenesis is to produce non-integrating lentiviral (NIL) vectors. NIL vectors have been produced by introducing combinations of mutations made to disable the integrase protein itself or to alter the integrase recognition sequences (aft) in the viral LTR (see e.g., Yanez-Munoz et al. (2006) Nat Med 12(3):348-353; Nightingale et al. (2006) Mol Ther 13(6):1121-1132). Recent in vitro and in vivo studies show that NIL vectors can mediate stable transduction and allow for high levels of transgene expression (see e.g., Yanez-Munoz et al. (2006) Nat Med 12(3):348-353; Nightingale et al. (2006) Mol Ther 13(6):1121-1132). Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. However, the vector forms generated by the above approaches comprise linear as well as three types of circular episomal vector forms. The presence of the linear DNA forms, which constitute the majority the episomal forms, carry the potential of inducing DNA damage response due to the presence of free double stranded DNA ends.
The presently disclosed subject matter provides improved integration-defective vector systems whose end product is a homogenous population of circular vector forms containing 1-LTR that are capable of mediating gene transfer into animal cells with a decreased risk of inducing DNA damage response. The fact that only 1-LTR forms are generated allows for better control of the non-integrating vectors. In addition, the new integration-defective vector system is significantly more efficient than the currently used non-integrating retroviral system in the context of particular applications, which are based on circular vector forms including but not limited to FLIP and cre mediated vector integration.